Microparticle-based transfection and activation of dendritic cells

ABSTRACT

The present invention provides an effective method for the transfection of dendritic cells by non-viral methods. The present invention provides this benefit by incubating dendritic cells and a specified transfection agent. The transfection agent comprises a polynucleotide and microparticles, with the microparticles being comprised of biodegradable polymer and cationic detergent. The dendritic cells and transfection agent are incubated for a time sufficient to transfect the dendritic cells with the polynucleotide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to Patent Application Ser. 60/146,391, filedJul. 29, 1999. This application is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods of providingdendritic cells for immunotherapy in connection with, for example,viruses or tumors. In particular, the invention relates to methods forgenerating antigen presenting dendritic cells by transfection, allowingfor, e.g., the activation and expansion of large numbers of viral- ortumor-antigen-specific T cells for use in adoptive cellularimmunotherapy against viruses and tumors.

BACKGROUND OF THE INVENTION

The generation of an immune response involves the sensitization ofhelper (CD4+)(T_(H)) and cytotoxic (CD8+)(CTL) T cell subsets throughtheir interaction with antigen presenting cells. Antigen presentingcells express major histocompatibility (MHC)-class I or class IImolecules associated with antigenic fragments (i.e., specific amino acidsequences derived from an antigen which bind to MHC I and MHC II forpresentation on the cell surface). The MHC in humans is also referred toas the HLA (human leukocyte antigen) complex. The sensitized CD4+ Tcells produce lymphokines that participate in the activation of B cellsas well as various T cell subsets. The sensitized CD8+ T cells increasein numbers in response to lymphokines and act to destroy cells thatexpress the specific antigenic fragments associated with matchingMHC-encoded class I molecules. In the course of a tumor or viralinfection, cytotoxic T cells eradicate cells expressing tumor or virusassociated antigens.

Dendritic cells (DCs) are thought to be the most potent antigenpresenting cells of the immune system (reviewed in Steinman, R. M. 1991.The dendritic cells system and its role in immunogenicity. Ann. Rev.Immunol. 9:271; Banchereau, J. B. and R. M. Steinman. 1998. Dendriticcells and the control of immunity. Nature. 392:245). Given their broadspectrum of roles in initiating the immune response by internalizing andprocessing antigens, migrating to lymphoid organs, secreting cytokines,and expressing co-stimulatory molecules required for lymphocytesignaling, it is no surprise that dendritic cells are logical targetsfor clinical use (Banchereau, J. B. and R. M. Steinman. 1998. Dendriticcells and the control of immunity. Nature. 392:245). By targetingantigens into dendritic cells in vivo or exposing dendritic cells toantigen ex vivo, it may be possible to enhance the immunogenicity ofvaccines by eliciting helper and cytotoxic T cells, antibodies, andIL-12 for prophylactic applications, or induce T cell mediatedanti-tumor responses for cancer immunotherapy. Akbari, et al. havesuggested that transfection and activation of dendritic cells are keyevents for immunity following DNA vaccination by scarification of theear skin in mouse models (O. Akbari, N. P., S. Garcia, R. Tascon, D.Lowrie, and B. Stockinger. 1999. DNA vaccination: transfection andactivation of dendritic cells as key events for immunity. J. Exp. Med.189:169). Anti-tumor CTL activity and protection against lethal tumorchallenge in mouse models have been demonstrated using cytokine-drivenbone-marrow-derived dendritic cells (BMDCs) pulsed with tumor-associatedpeptides (J. I. Mayordomo, T. Z., W. J. Storkus. 1995. Bonemarrow-derived dendritic cells pulsed with synthetic tumour peptideselicit protective and therapeutic antitumour immunity. Nature Med.1:1297), and whole tumor lysates (R. C. Fields, K. S., and J. J. Mule'.1998. Murine dendritic cells pulsed with whole tumor lysates mediatepotent antitumor immune responses in vitro and in vivo. Proc. Natl.Acad. Sci. USA 95:9482) transferred by the subcutaneous route.

In vitro generation of dendritic cells has been optimized sufficientlyso that genetic immunotherapy based on passive transfer of dendriticcells has become an attractive target for development (N. Romani, S. G.,D. Brang. 1994. Proliferating dendritic cell progenitors in human blood.J. Exp. Med. 180:83). However, in vitro transfection efficiency ofdendritic cells by non-viral methods has been extremely poor (J. F.Arthur, L. H. B., M. D. Roth, L. A. Bui, S. M. Kiertscher, R. Lau, S.Dubinett, J. Glaspy, W. H. McBride, and J. S. Economou. 1997. Acomparison of gene transfer methods in human dendritic cells. CancerGene Ther. 4:17) and has limited progress toward effectivedendritic-cell-based immunotherapy. While progress has been made by theuse of electroporation, the efficiency of transfection is extremely lowand results in substantial loss of cell viability (V. F. I. VanTendeloo, H.-W. S., F. Lardon, GLEE Vanham, G. Nijs, M. Lenjou, L.Hendriks, C. Van Broeckhoven, A. Moulijn, I. Rodrigus, P. Verdonk, D. R.Van Bockstaele, and Z. N. Berneman. 1988. Nonviral transfection ofdistinct types of human dendritic cells: high efficiency gene transferby electroporation into hematopoietic progenitor—but notmonocyte-derived dendritic cells. Gene Ther. 5:700). To date, no purelychemical method has been shown to be effective.

Particulate carriers have been used in order to achieve controlled,parenteral delivery of therapeutic compounds. Such carriers are designedto maintain the active agent in the delivery system for an extendedperiod of time. Examples of particulate carriers include those derivedfrom polymethyl methacrylate polymers, as well as microparticles derivedfrom poly(lactides) (see, e.g., U.S. Pat. No. 3,773,919),poly(lactide-co-glycolides), known as PLG (see, e.g., U.S. Pat. No.4,767,628) and polyethylene glycol, known as PEG (see, e.g., U.S. Pat.No. 5,648,095). Polymethyl methacrylate polymers are nondegradable whilePLG particles biodegrade by random nonenzymatic hydrolysis of esterbonds to lactic and glycolic acids, which are excreted along normalmetabolic pathways.

For example, U.S. Pat. 5,648,095 describes the use of microspheres withencapsulated pharmaceuticals as drug delivery systems for nasal, oral,pulmonary and oral delivery. Slow-release formulations containingvarious polypeptide growth factors have also been described. See, e.g.,International Publication WO 94/12158, U.S. Pat. 5,134,122 andInternational Publication WO 96/37216.

Particulate carriers have also been used with adsorbed or entrappedantigens in attempts to elicit adequate immune responses. Such carrierspresent multiple copies of a selected antigen to the immune system andpromote trapping and retention of antigens in local lymph nodes. Theparticles can be phagocytosed by macrophages and can enhance antigenpresentation through cytokine release. For example, commonly owned,co-pending application Ser. 09/015,652, filed Jan. 29, 1998, describesthe use of antigen-adsorbed and antigen-encapsulated microparticles tostimulate cell-mediated immunological responses, as well as methods ofmaking the microparticles.

In commonly owned provisional Patent Application 60/036,316, forexample, a method of forming microparticles is disclosed which comprisescombining a polymer with an organic solvent, then adding an emulsionstabilizer, such as polyvinyl alcohol (PVA), then evaporating theorganic solvent, thereby forming microparticles. The surface of themicroparticles comprises the polymer and the stabilizer. Polynucleotidessuch as DNA, polypeptides, and antigens may then be adsorbed on thosesurfaces. See also PCT US99/17308.

Commonly owned Provisional Application 60/146,391 discloses a method offorming microparticles with adsorbent surfaces that are capable ofadsorbing a variety of macromolecules including polynucleotides. In oneembodiment, the microparticles are comprised of both a polymer and adetergent. The microparticles are derived from a polymer, such as apoly(α-hydroxy acid), preferably, a poly(D,L-lactide-co-glycolide), apolyhydroxy butyric acid, a polycaprolactone, a polyorthoester, apolyanhydride, a polycyanoacrylate, and the like, and are formed withdetergents, such as cationic, anionic, or nonionic detergents, whichdetergents may be used in combination. Cationic detergents disclosed arecetrimide (CTAB), benzalkonium chloride, DDA (dimethyl dioctodecylammonium bromide), DOTAP, and the like. It is noted that thesemicroparticles yield improved adsorption of viral antigens, and providefor superior immune responses, as compared to microparticles formed by aprocess using only PVA.

Dendritic cells can capture antigen at peripheral sites viamacropinocytosis using membrane ruffling, or may also internalizeantigen by receptor-mediated processes involving FcγIII, the mannosereceptor, or the C-type lectin DEC-205 (reviewed in Lanzavecchia, A.1996. Mechanisms of antigen uptake for presentation. Curr. Op. Immunol.8:348). Thus, dendritic cells may be targeted by the capture of larger(>250 nm) particulate antigens by phagocytosis. Biodegradable polymermicrospheres such as poly-lactide-co-glycolide (PLG) are readilyinternalized by phagocytic cells up to a diameter of 5 μm (Ikada, Y. T.et al. 1990. Phagocytosis of polymer microspheres by macrophages. Adv.Polymer. Sci. 94:107) and have been utilized as carriers for drugdelivery systems.

Recently, Newman, et al. reported cytoplasmic delivery of Texas redlabeled dextran encapsulated in PLGA microspheres following phagocytosisin mouse peritoneal macrophages (K. D. Newman, G. K., J. Miller, V.Chlumecky, J. Samuel. 1999. Cytoplasmic delivery of a fluorescent probeby poly(D,L lactic-co-glycolic acid) microspheres. In 1999 AAPS AnnualMeeting Abstracts Online, vol. 1).

The application of synthetic biopolymers for nucleic acid delivery hasproven advantageous by protecting DNA against nuclease degradation andincreasing cellular uptake (C. Chavany, T. S.-B., T. Le Doan, F.Puisieux, P. Couvreur, and C. Helene. 1994. Adsorption ofoligonucleotides onto polyisohexylcyanoacrylate nanoparticles protectsthem against nucleases and increases their cellular uptake. Pharm. Res.11:1370).

Evidence for direct transfection of non professional antigen presentingcells mediated by PLG was recently reported by Ciftci and Su who foundPLG microparticles containing a DNA:polycation complex providedcontrolled release of DNA and surfactant-enhanced uptake and geneexpression in 293 and MCF-7 cells (K. Ciftci, J. S. 1999. DNA-PLGAmicroparticles: a promising delivery system for cancer gene therapy. In1999 AAPS Annual Meeting Abstracts Online, vol. 1).

While polyalkylcyanoacrylate nanoparticles have been used to bindCTAB-oligonucleotide complexes to deliver antisense oligonucleotides tomacrophage cell lines in vitro (C. Chavany, T. S.-B., T. Le Doan, F.Puisieux, P. Couvreur, and C. Helene. 1994. Adsorption ofoligonucleotides onto polyisohexylcyanoacrylate nanoparticles protectsthem against nucleases and increases their cellular uptake. Pharm. Res.11:1370; E. Fattal, C. V., I. Aynie, Y. Nakada, G. Lambert, C. Malvy,and P. Couvreur. 1998. Biodegradable polyalkylcyanoacrylatenanoparticles for the delivery of oligonucleotides. J. ControlledRelease 53:137), these vehicles have not been shown to transfectdendritic cells with plasmids carrying recombinant genes.

Hence, there is a need in the art for an effective non-viral techniquefor the transfection of dendritic cells. While microparticle technologyhas been heretofore used for introduction of polynucleotides into cells,applicants are aware of no such technology having been used for thetransfection of dendritic cells, which are notoriously resistant totransfection.

SUMMARY OF THE INVENTION

The present invention provides an effective method for the transfectionof dendritic cells by non-viral methods. The present invention providesthis benefit by incubating dendritic cells and a specified transfectionagent. The transfection agent comprises polynucleotide andmicroparticles, with the microparticles being comprised of abiodegradable polymer and a cationic detergent. The dendritic cells andtransfection agent are incubated for a time sufficient to transfect thedendritic cells with the polynucleotide.

For the transfecting agent, the cationic detergent preferably comprisesCTAB or cetrimide, while the polymer preferable is a poly(α-hydroxyacid), for example, a poly(lactide), a copolymer of D,L-lactide andcaprolactone, or a copolymer of D,L-lactide and glycolide or glycolicacid, such as poly(D,L-lactide-co-glycolide). In a further preferredembodiment, the polynucleotide is provided in the form of a plasmid. Instill further preferred embodiments, the polynucleotide encodes anantigen associated with a virus, such as HIV, meningitis A, meningitis Bor meningitis C, or a tumor.

The dendritic cells can originate from any available source, forexample, the bone marrow or blood of a vertebrate subject, preferably ahuman subject. Dendritic cells can be cultured, for example, for about 5to about 10 days prior to transfection, in the presence of appropriategrowth factors, for example, GM-CSF.

The dendritic cells and transfecting agent are preferably incubated forabout 24 hours under appropriate conditions.

In some embodiments of the present invention, an effective amount of thetransfected dendritic cells of the present invention are administered toa vertebrate subject in need thereof. In other embodiments, T cells arefirst activated by the dendritic cells of the present invention and thenadministered to a vertebrate subject in need thereof. The dendriticcells and/or T cells may originate, for example, from the vertebratesubject or a healthy vertebrate subject MHC-matched to the vertebratesubject. The dendritic cells and or T cells may be administeredparenterally to the vertebrate subject.

One advantage of the present invention is that polynucleotides can beefficiently internalized by dendritic cells.

Another advantage of the present invention is that gene expression canbe effected within dendritic cells.

Yet another advantage of the present invention is that antigen can beprocessed and presented in connection with MHC molecules on the surfaceof dendritic cells.

Another advantage of the present invention is that polynucleotides canbe rapidly internalized and expressed, with antigen presentation.

Still another advantage of the present invention is that the methods ofthe invention can be used, for example, in genetic immunotherapy orvaccination with relative safely. For instance, both cationicdetergents, such as CTAB, and biodegradable polymers, such as PLG, havebeen utilized in biomedical applications. Moreover, the obvious safetyconcerns with the use of live viral vectors can be avoided (reviewed inRock, S. R. et al. 1998. Fully mobilizing host defense: building bettervaccines. Nature Biotech. 16:1025).

These and other embodiments and advantages will become readily apparentto those skilled in the art upon review of this specification and theclaims set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Agarose gel electrophoresis of RT-PCR products for detection oftarget gene expression. Lane designations are as follows: 1) 500 bp DNAladder, 2-4) β-actin control RT-PCR reactions from untreated, plasmidDNA, and PLG-CTAB-DNA treated bone-marrow-derived dendritic cells(BMDCs), 5) 100 bp DNA ladder, 6) untreated mRNA prep with control spikeof pCMV-gag DNA, 8-10) p55gag RT-PCR from untreated, plasmid DNA, andPLG-CTAB-DNA treated BMDCs, 11-13) PCR negative control from untreated,plasmid DNA, and PLG-CTAB-DNA treated BMDCs, 14) pCMV-gag DNA PCRpositive control.

FIG. 2 illustrates IL-2 production level after stimulation of an MHCclass I T cell hybridoma with bone-marrow-derived dendritic cells(BMDCs). Both immature (6 day) and mature (9 day) BMDCs were examined.BMDC treatment within each maturity group is as follows (from left toright): untreated, treatment with PLG-CTAB, treatment withPLG-CTAB-pCMVgag DNA, treatment with PLG-CTAB-luc DNA, treatment withnaked pCMVgag DNA, and treatment with naked luc DNA.

FIG. 3 illustrates IL-2 production level after stimulation of T cellhybridoma (left y-axis, bar series) with BMDCs that were incubated withvarying concentrations of pCMV-gag plasmid DNA formulated on PLG-CTABmicroparticles. FIG. 3 also illustrates % viability (right y-axis, lineseries) of BMDCs that were incubated with varying concentrations ofpCMV-gag plasmid DNA formulated on PLG-CTAB microparticles. Whereappropriate, data points represent the average values and standard errorof duplicate samples.

FIG. 4 illustrates IL-2 production level after stimulation ofgag-specific T cell hybridoma with either naïve (untreated) BMDCs orBMDCs treated with PLG-CTAB-pCMVgag DNA, and after pulsing with anexcess of synthetic peptide epitope. Varying ratios of T cells toantigen presenting cells (i.e., BMDCs) were studied, with the number ofT cells being held constant in all cases. Data points represent themean±error of duplicate samples assayed by a series of dilutions.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, polymer chemistry,biochemistry, molecular biology, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton,Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowickand N. Kaplan, eds., Academic Press, Inc.); Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Handbook of Surface andColloidal Chemistry (Birdi, K. S., ed, CRC Press, 1997) andSeymour/Carraher's Polymer Chemistry (4th edition, Marcel Dekker Inc.,1996).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The term “dendritic cells” is used herein to refer to antigen presentingcells characterized by their peculiar dendritic morphology and multiplethin-membrane projections, and by their high density of class II MHCmolecules. Dendritic cells include Langerhans cells of the skin, “veiledcells” of afferent lymphatics, follicular dendritic cells, dendriticcells of the spleen, and interdigitating cells of lymphoid organs.Dendritic cells can be obtained from the skin, spleen, bone marrow,lymph nodes, other lymphoid organs, and peripheral blood cord blood.Preferably, dendritic cells are obtained from blood or bone marrow foruse in the invention.

The term “microparticle” as used herein, refers to a particle of about100 nm to about 150 μm in diameter, more preferably about 200 nm toabout 30 μm in diameter, and most preferably about 500 nm to about 10 μmin diameter. Microparticle size is readily determined by techniques wellknown in the art, such as photon correlation spectroscopy, laserdiffractometry and/or scanning electron microscopy.

Microparticles for use herein are preferably formed from materials thatare preferable sterilizable, non-toxic and biodegradable. Such materialsinclude, without limitation, poly(α-hydroxy acid), polyhydroxybutyricacid, polycaprolactone, polyorthoester, polyanhydride, PACA, andpolycyanoacrylate. Preferably, microparticles for use with the presentinvention are derived from a poly(α-hydroxy acid), in particular, from apoly(lactide) (“PLA”) or a copolymer of D,L-lactide and glycolide orglycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or“PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, for example, inthe case of the copolymers such as PLG, a variety of co-monomer(lactide:glycolide) ratios.

The term “cationic detergent” as used herein includes cationicsurfactants and emulsion stabilizers. Cationic detergents include, butare not limited to, cetrimide, CTAB, benzalkonium chloride, DDA(dimethyl dioctodecyl ammonium bromide),Dioleoyl-3-Trimethylammonium-Propane (DOTAP), and the like.

A “polynucleotide” is a nucleic acid polymer. Polynucleotides accordingto the present invention are preferably of the minimum transfection unitlength, which is on the order of about 1 kb. Furthermore, a“polynucleotide” can include both double- and single-stranded sequences,and can include naturally derived and synthetic DNA sequences. The termalso includes sequences that include any of the known base analogs ofDNA and RNA, and includes modifications, such as deletions, additionsand substitutions (generally conservative in nature) to nativesequences.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include modifications,such as deletions, additions and substitutions (generally conservativein nature), to native sequence.

By “antigen” is meant a molecule that contains one or more epitopescapable of stimulating an immunological response when the antigen ispresented on a dendritic cell surface in accordance with the presentinvention. Normally, an epitope will include between about 3-15,generally about 5-15, amino acids. Epitopes of a given protein can beidentified using any number of epitope mapping techniques, well known inthe art. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.For example, linear epitopes may be determined by, e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra.

For purposes of the present invention, antigens can be derived from anyof several known viruses, bacteria, parasites and fungi, as well as anyof the various tumors. Furthermore, for purposes of the presentinvention, an “antigen” refers to a protein, which includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature), to the native sequence, so long as the abilityto elicit an immunological response is maintained. These modificationsmay be deliberate, as through site-directed mutagenesis, or may beaccidental, such as through mutations of hosts that produce theantigens.

An “immunological response” or “immune response” is the development in asubject of a humoral and/or a cellular immune response to moleculespresent in the composition of interest. For purposes of the presentinvention, a “humoral immune response” refers to an immune responsemediated by antibody molecules, while a “cellular immune response” isone mediated by T-lymphocytes and/or other white blood cells. Thus, animmunological response as used herein may be one which stimulates theproduction of cytotoxic T cells, and/or the production or activation ofhelper T-cells. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

Vaccines and immunogenic compositions are both contemplated inconnection with the present invention.

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, mammals such as cattle, sheep, pigs,goats, horses, and humans; domestic animals such as dogs and cats; andbirds, including domestic, wild and game birds such as cocks and hensincluding chickens, turkeys and other gallinaceous birds. The term doesnot denote a particular age. Thus, both adult and newborn animals areintended to be covered.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

B. Formation of Microparticles

In the present invention, a polynucleotide comprising an antigen ofinterest is adsorbed upon microparticles formed from a polymer and acationic detergent.

The adsorption of polynucleotides to the surface of the adsorbentmicroparticles occurs via any bonding-interaction mechanism, including,but not limited to, ionic bonding, hydrogen bonding, covalent bonding,Van der Waals bonding, and bonding through hydrophilic/hydrophobicinteractions. Those of ordinary skill in the art may readily selectcationic detergents appropriate for the invention. As noted above, knowncationic detergents include, but are not limited to, cetyl trimethylammonium bromide (CTAB), cetrimide (a mixture consisting chiefly oftetradecyltrimethylammonium bromide, together with smaller amounts ofdodecyltrimethylammonium bromide and CTAB), benzalkonium chloride, DDA(dimethyl dioctodecyl ammonium bromide), DOTAP, and the like. CTAB isparticularly preferred. Microparticles manufactured with cationicdetergents, such as CTAB, e.g., CTAB-PLG microparticles, readily adsorbnegatively charged polynucleotides.

Biodegradable polymers for manufacturing microparticles for use with thepresent invention are readily commercially available from, e.g.,Boehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham,Ala. For example, useful polymers for forming the microparticles hereininclude those derived from polyhydroxybutyric acid; polycaprolactone;polyorthoester; polyanhydride; as well as a poly(α-hydroxy acid), suchas poly(L-lactide), poly(D,L-lactide) (both known as “PLA” herein),poly(hydroxybutyrate), copolymers of D,L-lactide and glycolide, such aspoly(D,L-lactide-co-glycolide) (designated as “PLG” or “PLGA” herein) ora copolymer of D,L-lactide and caprolactone. Particularly preferredpolymers for use herein are PLA and PLG polymers. These polymers areavailable in a variety of molecular weights, and the appropriatemolecular weight for a given use is readily determined by one of skillin the art. Thus, e.g., for PLA, a suitable molecular weight will be onthe order of about 2000 to 5000. For PLG, suitable molecular weightswill generally range from about 10,000 to about 200,000, preferablyabout 15,000 to about 150,000, and most preferably about 50,000 to about100,000.

If a copolymer such as PLG is used to form the microparticles, a varietyof lactide:glycolide ratios will find use herein. PLG copolymers withvarying lactide:glycolide ratios and molecular weights are readilyavailable commercially from a number of sources including fromBoehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham,Ala. These polymers can also be synthesized by simple polycondensationof the lactic acid component using techniques well known in the art,such as described in Tabata et al., J. Biomed. Mater. Res. (1988)22:837-858.

The polynucleotide/microparticles are prepared using any of severalmethods well known in the art. For example, double emulsion/solventevaporation techniques, such as those described in U.S. Pat. 3,523,907and Ogawa et al., Chem. Pharm. Bull. (1988) 36:1095-1103, can be usedherein to make the microparticles.

A water-in-oil-in-water (w/o/w) solvent evaporation system can be usedto form the microparticles, as described by O'Hagan et al., Vaccine(1993) 11:965-969 and Jeffery et al., Pharm. Res. (1993) 10:362. In thistechnique, the particular polymer is combined with an organic solvent,such as ethyl acetate, dimethylchloride (also called methylene chlorideand dichloromethane), acetonitrile, acetone, chloroform, and the like.The polymer will be provided in about a 1-30%, preferably about a 2-15%,more preferably about a 3-10% and most preferably, about a 4% solution,in organic solvent. The polymer solution is emulsified using, e.g., ahomogenizer. The emulsion is then optionally combined with a largervolume of an aqueous solution of an emulsion stabilizer such aspolyvinyl alcohol (PVA), polyvinyl pyrrolidone, and a detergent,specifically a cationic detergent. The emulsion may be combined withmore than one emulsion stabilizer and/or detergent, e.g., a combinationof PVA and a cationic detergent. Certain polynucleotides may adsorb morereadily to microparticles having a combination of stabilizers and/ordetergents. Where an emulsion stabilizer is used, it is typicallyprovided in about a 2-15% solution, more typically about a 4-10%solution. Generally, a weight-to-weight detergent to polymer ratio inthe range of from about 0.00001:1 to about 0.1:1 will be used, morepreferably from about 0.0001:1 to about 0.01:1, more preferably fromabout 0.001:1 to about 0.01:1, and even more preferably from about0.005:1 to about 0.01:1. The mixture is then homogenized to produce astable w/o/w double emulsion. Organic solvents are then evaporated.

The formulation parameters can be manipulated to allow the preparationof small microparticles on the order of 0.05 μm (50 nm) to largermicroparticles 50 μm or even larger. See, e.g., Jeffery et al., Pharm.Res. (1993) 10:362-368; McGee et al., J. Microencap. (1996). Forexample, reduced agitation results in larger microparticles, as does anincrease in internal phase volume. Small particles are produced by lowaqueous phase volumes with high concentrations of emulsion stabilizers.

Microparticles can also be formed using spray-drying and coacervation asdescribed in, e.g., Thomasin et al., J. Controlled Release (1996)41:131; U.S. Pat. 2,800,457; Masters, K. (1976) Spray Drying 2nd Ed.Wiley, New York; air-suspension coating techniques, such as pan coatingand Wurster coating, as described by Hall et al., (1980) The “WursterProcess” in Controlled Release Technologies: Methods, Theory, andApplications (A. F. Kydonieus, ed.), Vol. 2, pp. 133-154 CRC Press, BocaRaton, Fla. and Deasy, P. B., Crit. Rev. Ther. Drug Carrier Syst. (1988)S(2):99-139; and ionic gelation as described by, e.g., Lim et al.,Science (1980) 210:908-910.

Particle size can be determined by, e.g., laser light scattering, usingfor example, a spectrometer incorporating a helium-neon laser.Generally, particle size is determined at room temperature and involvesmultiple analyses of the sample in question (e.g., 5-10 times) to yieldan average value for the particle diameter. Particle size is alsoreadily determined using scanning electron microscopy (SEM).

Following preparation, microparticles can be stored as is orfreeze-dried for future use.

C. Isolation of Dendritic Cells

Dendritic cells are obtained from any tissue where they reside includingnon-lymphoid tissues such as the epidermis of the skin (Langerhanscells) and lymphoid tissues such as the spleen, bone marrow, lymph nodesand thymus as well as the circulatory system including blood (blooddendritic cells), for example peripheral blood and cord blood, and lymph(veiled cells).

For example, explants of mouse (Larsen et al., J. Exp. Med.172:1483-1493 (1990)) or human skin (Richters et al., J. Invest.Dermatol. (1994)) placed in organ culture permit selective migration ofdendritic cells into the medium surrounding the explant.

Recent studies have described methods for the isolation and expansion ofhuman dendritic cells, including, from human peripheral blood.(Macatonia et al., 1991, Immunol. 74: 399-406; O'Doherty et al., 1993,J. Exp. Med. 178: 1067-1078 (isolation); and Markowicz et al., 1990, J.Clin. Invest. 85: 955-961; Romani et al., 1994, J. Exp. Med. 180: 83-93;Sallusto et al., 1994, J. Exp. Med. 179: 1109-1118; Berhard et al.,1995, J. Exp. Med. 55: 1099-1104 (expansion)).

Van Tendeloo et al., 1998, Gene Ther. 5: 700-707, discloses techniquesfor deriving dendritic cells (including Langerhans' cells) from CD34+progenitor cells obtained from bone marrow and cord blood and frommononuclear cells from peripheral blood.

Dendritic cells may also be treated to induce maturation or activation,e.g., by culturing, preferably in the presence of a specific growth orstimulatory factor or factors. In the examples below, dendritic cellsare modified by culturing with GM-CSF.

Additional techniques relating to the preparation of dendritic cells canbe found, for example, in U.S. Pat. Nos. 5,788,963, 5,962,318, and5,851,756, the disclosures of which are herein incorporated byreference.

According to a preferred embodiment of the invention, dendritic cellsare obtained from a patient to be treated. The dendritic cells are usedto activate T cells of the patient, either in vitro or in vivo, forimmunotherapy.

According to an alternate embodiment, dendritic cells are obtained froma healthy individual. The relevant HLA antigens (both class I and II,e.g., HLA-A, B, C and DR), for example, on the individual's peripheralblood mononuclear cells (PBMC's), are identified and dendritic cellsthat match the patient, in terms of HLA antigens, are isolated andexpanded as described above. For example, in certain instances, a latestage cancer patient who has been treated with radiation and/orchemotherapy agents is not able to provide sufficient or efficientdendritic cells. Thus, dendritic cells from healthy HLA-matchedindividuals, such as siblings, can be obtained and expanded using any ofthe methods described above.

D. Antigens

Selected antigens that may be expressed include one or more selectedantigens of a vertebrate infectious agent or cancer and can correspondto either structural or non-structural proteins. The invention hereindescribed can provide for association of such antigens with MHCmolecules at the surface of dendritic cells such that an immune responseto the antigen of interest can be mounted.

For example, the present invention is useful for stimulating an immuneresponse against a wide variety of antigens from the herpes virusfamily, including proteins derived from herpes simplex virus (HSV) types1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigensderived from varicella zoster virus (VZV), Epstein-Barr virus (EBV) andcytomegalovirus (CMV) including CMV gB and gH; and antigens derived fromother human herpesviruses such as HHV6 and HHV7. (See, e.g. Chee et al.,Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp.125-169, for a review of the protein coding content of cytomegalovirus;McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a discussion ofthe various HSV-1 encoded proteins; U.S. Pat. 5,171,568 for a discussionof HSV-1 and HSV-2 gB and gD proteins and the genes encoding therefor;Baer et al., Nature (1984) 310:207-211, for the identification ofprotein coding sequences in an EBV genome; and Davison and Scott, J.Gen. Virol. (1986) 67:1759-1816, for a review of VZV.)

Antigens from the hepatitis family of viruses, including hepatitis Avirus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV), can also be conveniently used in the techniques described herein.By way of example, the viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodesseveral viral proteins, including E1 (also known as E) and E2 (alsoknown as E2/NSI) and an N-terminal nucleocapsid protein (termed “core”)(see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion ofHCV proteins, including E1 and E2). Each of these proteins, as well asantigenic fragments thereof, will find use in the present compositionand methods.

Similarly, the sequence for the δ-antigen from HDV is known (see, e.g.,U.S. Pat. 5,378,814) and this antigen can also be conveniently used inthe present composition and methods. Additionally, antigens derived fromHBV, such as the core antigen, the surface antigen, sAg, as well as thepresurface sequences, pre-S1 and pre-S2 (formerly called pre-S), as wellas combinations of the above, such as sAg/pre-S1, sAg/pre-S2,sAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See, e.g.,“HBV Vaccines—from the laboratory to license: a case study” in Mackett,M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176,for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840,5,098,704, 5,324,513, incorporated herein by reference in theirentireties; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum etal., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991)65:5457-5464.

Antigens derived from other viruses will also find use in the claimedcompositions and methods, such as without limitation, proteins frommembers of the families Picornaviridae (e.g., polioviruses, etc.);Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.);Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae(e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumpsvirus, measles virus, respiratory syncytial virus, etc.);Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.);Bunyaviridae; Arenaviridae; Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1(also known as HTLV-III, LAV, ARV, hTLR, etc.)), including but notlimited to antigens from the isolates HIV_(IIIb), HIV_(SF2), HIV_(LAV),HIV_(LAI), HIV_(MN)); HIV-1_(CM235), HIV-1_(US4); HIV-2; simianimmunodeficiency virus (SIV) among others. Additionally, antigens mayalso be derived from human papillomavirus (HPV) and the tick-borneencephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed.1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,eds. 1991), for a description of these and other viruses.

More particularly, the gp120 envelope proteins from any of the above HIVisolates, including members of the various genetic subtypes of HIV, areknown and reported (see, e.g., Myers et al., Los Alamos Database, LosAlamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al.,Human Retroviruses and Aids, 1990, Los Alamos, N. Mex.: Los AlamosNational Laboratory; and Modrow et al., J. Virol. (1987) 61:570-578, fora comparison of the envelope sequences of a variety of HIV isolates) andantigens derived from any of these isolates will find use in the presentmethods. Furthermore, the invention is equally applicable to otherimmunogenic proteins derived from any of the various HIV isolates,including any of the various envelope proteins such as gp160 and gp41,gag antigens such as p24gag and p55gag, as well as proteins derived fromthe pol region.

Influenza virus is another example of a virus for which the presentinvention will be particularly useful. Specifically, the envelopeglycoproteins HA and NA of influenza A are of particular interest forgenerating an immune response. Numerous HA subtypes of influenza A havebeen identified (Kawaoka et al., Virology (1990) 179:759-767; Webster etal., “Antigenic variation among type A influenza viruses,” p. 127-168.In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses.Springer-Verlag, New York). Thus, proteins derived from any of theseisolates can also be used in the compositions and methods describedherein.

Antigens derived from meningitis A, meningitis B, meningitis C, andother related viruses will also find use in the compositions and methodsof the present invention. For examples of meningitis B antigens see, forexample, PCT 99/00695 filed Apr. 7, 1999; PCT IB98/01665 filed Oct. 9,1998 and PCT US99/09346 filed Apr. 30, 1999.

Non-viral organisms that are controlled by T cell immune responsesinclude: pathogenic protozoa (e.g. Pneumocystis carinii, Trypanosoma,Leishmania, Plasmodia, and Toxoplasma gondii); bacteria (e.g.,Mycobacteria, and Legioniella) and fungi (e.g. Histoplasma capsulatumand Cocidioides immitus). Hence, antigens derived from these organismsare also useful in connection with the present invention.

Tumor antigens for use in the invention include, but are not limited to,melanoma tumor antigens (Kawakami et al., Proc. Natl. Acad. Sci. USA91:3515-3519 (1994); Kawakami et al., J. Exp. Med., 180:347-352 (1994);Kawakami et al. Cancer Res. 54:3124-3126 (1994), including MART-1(Coulie et al., J. Exp. Med. 180:35-42 (1991), gp100 (Wick et al., J.Cutan. Pathol. 4:201-207 (1988) and MAGE antigen, MAGE-1, MAGE-2 andMAGE-3 (Van der Bruggen et al., Science, 254:1643-1647 (1991)); CEA,TRP-1, P-15 and tyrosinase (Brichard et al., J. Exp. Med. 178:489(1993)); HER-2/neu gene product (U.S. Pat. 4,968,603); estrogenreceptor, milk fat globulin, p53 tumor suppressor protein (Levine, Ann.Rev. Biochem. 62:623 (1993)); mucin antigens (Taylor-Papdimitriou,International Pub. WO90/05142)); telomerases; nuclear matrix proteins;prostatic acid phosphatase; papilloma virus antigens; and antigensassociated with the following cancers: melanomas, metastases,adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias,uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, bladder cancer, kidney cancer, pancreatic cancer and others(e.g., Rosenberg, Ann. Rev. Med. 47:481-91 (1996).

E. Polynucleotides

In accordance with the invention, one or more polynucleotides areinserted ex vivo into dendritic cells, such that one or more selectedantigens are presented in effective amounts on the surface of thedendritic cells. By “effective amount” is meant that presentation issufficient to enable the dendritic cells to provoke an immune response.

Techniques for nucleic acid manipulation are well known. Reagents usefulin applying such techniques, such as restriction enzymes and the like,are widely known in the art and commercially available from a number ofvendors.

Large amounts of polynucleotide sequences encoding the selected antigensfor expression in the dendritic cells of the invention may be obtainedusing known procedures for molecular cloning and replication of a vectorcarrying the sequences in a suitable host cell. The nucleic acidsequences for use in the present invention may also be produced in partor in total by chemical synthesis, and may be performed on commercialautomated oligonucleotide synthesizers.

Polynucleotides encoding the desired antigens for presentation in thedendritic cells are preferably recombinant expression vectors in whichhigh levels of expression may occur, and which contain appropriateregulatory sequences for transcription and translation of the insertednucleic acid sequence. The vectors may also contain polynucleotidesequences encoding selected class I and class II MHC molecules,costimulation and other immunoregulatory molecules, ABC transporterproteins, including the TAP1 and TAP2 proteins. Thus, variouscombinations of polynucleotide sequences may be inserted in a suitableexpression vector or vectors. The vector may contain additional elementsneeded for subsequent replication, such as an origin of replication. Thevectors may also contain at least one positive marker that enables theselection of dendritic cells carrying the inserted nucleic acids.

Preferred recombinant expression vectors for the invention includeplasmid vectors. Preferred plasmid expression vectors include pCMV (see,for example, U.S. Pat. 5,688,688, the entire disclosure of which ishereby incorporated by reference).

Polynucleotides encoding the desired antigen or antigens are introducedinto dendritic cells using the transfection methods of the presentinvention discussed below.

F. Association of Microparticles with Polynucleotides

In order to associate a polynucleotide of interest with a microparticleof interest, microparticles are simply mixed with polynucleotides, forexample, in an appropriate buffer solution. The resulting formulationcan be lyophilized prior to use. Generally, polynucleotides are added tothe microparticles to yield microparticles with adsorbed polynucleotideshaving a weight-to-weight ratio of from about 0.0001:1 to 0.25:1polynucleotides to microparticles, preferably, 0.001:1 to 0.1, morepreferably 0.01 to 0.05. Polynucleotide content of the microparticlescan be determined using standard techniques.

The microparticles of the present invention may have polynucleotidesentrapped or encapsulated within them, as well as having polynucleotidesadsorbed thereon.

The association of the microparticle with the polynucleotide is referredto alternatively herein as “polynucleotide/microparticles”,“transfecting agent” and “transfection agent”.

G. Transfection of Dendritic Cells

Once the dendritic cells and polynucleotide/microparticles are prepared,they are incubated in solution for a time and at a temperaturesufficient for transfection to occur. According to a preferredembodiment, dendritic cells and polynucleotide/microparticles areincubated for 24 hours at 37° C. in humidified CO₂ incubator.

Expression of the polynucleotide of interest after transfection intodendritic cells may be confirmed by immunoassays or biological assays.For example, expression of introduced polynucleotides into cells may beconfirmed by detecting the binding to the cells of labeled antibodiesspecific for the antigens of interest using assays well known in the artsuch as FACS (Fluorescent Activated Cell Sorting) or ELISA(enzyme-linked immunoabsorbent assay) or by simply by staining (e.g.,with β-gal) and determining cell counts.

T cell activation may be detected by various known methods, includingmeasuring changes in the proliferation of T cells, killing of targetcells and secretion of certain regulatory factors, such as lymphokines,expression of mRNA of certain immunoregulatory molecules, or acombination of these.

H. Use of Dendritic Cells to Present Antigen In Vitro and In Vivo

According to an embodiment of the invention, dendritic cells transfectedby polynucleotide/microparticles using any of the methods describedherein are used to activate T cells in vitro. T cells or a subset of Tcells can be obtained from various lymphoid tissues. Such tissuesinclude but are not limited to spleens, lymph nodes, and peripheralblood.

The cells can be co-cultured with transfected dendritic cells as a mixedT cell population or as a purified T cell subset. For instance, it maybe desired to culture purified CD8+ T cells with antigen transfecteddendritic cells, as early elimination of CD4+ T cells may prevent theovergrowth of CD4+ cells in a mixed culture of both CD8+ and CD4+ Tcells. T cell purification may be achieved by positive or negativeselection, including but not limited to, the use of antibodies directedto CD2, CD3, CD4, CD5, and CD8. On the other hand, it may be desired touse a mixed population of CD4+ and CD8+ T cells to elicit a specificresponse encompassing both a cytotoxic and T_(H) immune response.

After activation in vitro, the T cells are administered to a patient ina dose sufficient to induce or enhance an immune response to theselected antigen expressed by the dendritic cells of the invention.

T cells, as well as dendritic cells as described below, may beintroduced into the subject to be treated by using one of a number ofmethods of administration of therapeutics known in the art. For example,the cells may be administered (with or without adjuvant) parenterally(including, for example, intravenous, intraperitoneal, intramuscular,intradermal, and subcutaneous administration). Alternatively, the cellsmay be administered locally by direct injection into a tumor or infectedtissue. Adjuvants include any known pharmaceutically acceptable carrier.Parenteral vehicles for use as pharmaceutical carriers include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride, andlactated Ringer's. Other adjuvants may be added as desired such asantimicrobials.

As an example, T cells may be administered, by intravenous infusion, atdoses of about 10⁸ to 10⁹ cells/m² of body surface area (see, Ridell etal., 1992, Science 257: 238-241). Infusion can be repeated at desiredintervals, for example, monthly. Recipients are monitored during andafter T cell infusions for any evidence of adverse effects.

According to a preferred embodiment, the T cells are obtained from thesame patient from whom the dendritic cells were obtained.

According to another embodiment, the T cells are obtained from a patientand the dendritic cells, which are used to stimulate the T cells, areobtained from an HLA-matched healthy donor (e.g., a sibling), or viceversa.

According to yet another embodiment, both the T cells and the dendriticcells are obtained from an HLA-matched healthy donor. This embodimentmay be particularly advantageous, for example, when the patient is alate stage cancer patient who has been treated with radiation and/orchemotherapy agents and may not be able to provide sufficient orefficient dendritic or T cells.

According to another embodiment of the invention, dendritic cellsisolated from a patient are cultured, transfected in vitro andadministered back to the patient to stimulate an immune response,including T cell activation. As such, the dendritic cells constitute avaccine and/or immunotherapeutic agent. As an example, dendritic cellspresenting antigen are administered, via intravenous infusion, at a doseof, for example, about 10⁶ to 10⁸ cells. The immune response of thepatient can be monitored. Infusion can be repeated at desired intervalsbased upon the patient's immune response.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLES Example 1

Plasmids and DNA formulations. pCMVgag plasmid encoding HIV p55 gagprotein under the control of the cytomegalovirus early promoter waspurified by ion-exchange chromatography using an Qiagen Endo Free GigaKit and determined to be endotoxin free (<2.5 EU/ml). For uptake andreporter gene expression experiments, a rhodamine PNA-clamp plasmidencoding B-galactosidase was purchased from Gene Therapy Systems (SanDiego, Calif.).

Cationic microparticles were prepared using a modified solventevaporation process. The microparticles were prepared by emulsifying 10ml of a 5% w/v polymer (RG 504 PLG (Boehringer Ingelheim)) solution inmethylene chloride with 1 ml of PBS (Phosphate-Buffered Saline) at highspeed using an IKA homogenizer. The primary emulsion was then added to50 ml of distilled water containing cetyl trimethyl ammonium bromide(CTAB) (0.5% w/v). This resulted in the formation of a w/o/w emulsion,which was stirred at 6000 rpm for 12 hours at room temperature, allowingthe methylene chloride to evaporate. The resulting microparticles werewashed twice in distilled water by centrifugation at 10,000 g and freezedried.

Plasmid DNA was adsorbed onto the microparticles targeting a 1% w/w load(by incubating 100 mg of cationic microparticles in a 1 mg/ml solutionof DNA at 4° C. for 6 hours). The particles were separated bycentrifugation, washed with TE buffer and lyophilized until use. Thesize distribution of the PLG-CTAB microparticles was determined using aparticle size analyzer (Malvern Instruments, U.K.); formulationsutilized in this study had a mean size of approximately 1 μm. Withoutwishing to be held to any particular theory of operation, the use of thecationic surfactant is believed to result in a net surface positivecharge for the adsorption of rhodamine-labeled plasmid DNA. Actual DNAload was quantified by assaying free DNA content in the supernatant andsubtracting from total input DNA. PLG-CTAB-DNA formulations utilized inthis example had an actual DNA load ranging from 0.64-0.81% (w/w).

Cell culture. All cells used in this study were cultured in RPMI-1640(BioWhittaker) supplemented with 10% heat-inactivated FBS, 2 mMglutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.05 mM2-mercaptoethanol at 37° C. in a humidified 7% CO₂ incubator. The murineT cell hybridoma 12.2 is an MHC class I, d-restricted line whichrecognizes the p7g peptide of HIV gag protein (provided by Gillis Otten,Chiron Corp.)

Bone marrow isolation. Female Balb/c mice, 6-8 weeks old, were obtainedfrom Charles River Laboratories (Holister, Calif.). Bone marrow wasflushed from the femurs and tibia, washed and frozen (−80° C.) inheat-inactivated fetal bovine serum supplemented with 10% cell-culturegrade DMSO (dimethyl sulfoxide) at a density of 2×10⁷ cells/ml.

Generation of bone-marrow-derived dendritic cells (BMDCs). Frozen cellaliquots were rapidly thawed and washed to remove DMSO. Cells wereplated in 150 mm suspension culture dishes containing 20 ml supplementedRPMI (see above) with the addition of 200 units/ml murine GM-CSF(Preprotech). On day 3 of culture, cells were again supplemented withmurine GM-CSF, and on day 5, one-half of the culture volume wascentrifuged to replace fresh medium containing GM-CSF. BMDCs wereharvested by gentle pipetting. Unless otherwise indicated, bone marrowderived dendritic cells were incubated with the gene-encoding antigen onday 6 and incubated 24 h further. BMDCs were analyzed for cell surfacemarkers by FACS (fluorescence-activated cell sorter) and werecharacterized as immature by staining positive for CD11c, CD11b,H-dK^(d), I-A^(d(low)), CD80^((low)), and CD86^((low)) (PharMingen).

Cellular uptake and fluorescence microscopy. BMDCs were plated at adensity of 1×10⁶ cells in 2 ml medium in 6-well culture dishes.Rhodamine-labelled DNA in the form of naked plasmid or formulated onPLG-CTAB microparticles was added to the wells at 1 μg DNA/ml. Followingovernight incubation, cells were washed and applied to Superfrostmicroscope slides (Fisher Scientific) by cytospin (4000 rpm×5 min).Slides were air-dried, mounted in Vectashield (Vector, Burlingame,Calif.) and visualized using a Zeiss Axiophot fluorescence microscopewith rhodamine filters (Chroma, Brattleboro, Vt.). Images weredocumented on Kodak EliteChrome film (100 ASA) and scanned into AdobePhotoshop.

Naked plasmid DNA was readily internalized into punctate arrangementssuggestive of endosomes. In contrast, the cellular distribution ofrhodamine-labeled plasmid DNA formulated on PLG-CTAB-DNA microparticlessuggested a more diffuse distribution of the rhodamine signal. Similarpatterns of internalization have been observed with DiI-labeledmicroparticles as well as PLG-CTAB microparticles containingencapsulated FITC-labeled bovine serum albumin. Without wishing to beheld to any particular theory, it appears as though the cationicsurfactant may disrupt the endosomal compartment allowing DNAlocalization to the nucleus.

Example 2

RNA isolation and RT-PCR. BMDCs were plated at a density of 0.5×10⁶cells/ml in RPMI+GM-CSF on day 6 of culture. Cells were either leftuntreated as negative control, or incubated in the presence of 1 μg/mlpCMV-gag DNA either alone (naked) or formulated on PLG-CTABmicrospheres. Following 24 h incubation, 2×10⁵ cells were removed,washed 2× in cold PBS (Life Technologies), then lysed per manufacturer'sinstructions for the mRNA Capture kit (Roche) and frozen at ±80° C.Samples were thawed on ice with the addition of RNase-free DNase andRNase inhibitor (Roche). The mRNA isolation protocol was then followedfor isolation of biotin-hybridized mRNA in streptavid in PCR tubes. ThePromega Reverse Transcription System (Madison, Wis.) was utilized forcDNA synthesis according to manufacturer's instructions, and thereaction was run at 45° C. for 45 min, followed by heat inactivation at99° C. for 5 min. PCR control tubes were treated as stated above butwithout the addition of AMV-reverse transcriptase for subsequentdetermination of the presence of contaminating plasmid DNA. For PCRamplification, samples were set up to amplify a 300 bp region of the HIVgag gene, or B-actin as a positive control using general PCR conditions.

Products were analyzed by agarose gel electrophoresis (FIG. 1). Lanedesignations are as follows: 1) 500 bp DNA ladder, 2-4) β-actin controlRT-PCR reactions from untreated, plasmid DNA treated, and PLG-CTAB-DNAtreated BMDCs, 5) 100 bp DNA ladder, 6) untreated mRNA prep with controlspike of pCMV-gag DNA, 8-10) p55gag RT-PCR from untreated, plasmid DNAtreated, and PLG-CTAB-DNA treated BMDCs, 11-13) PCR negative controlfrom untreated, plasmid DNA treated, and PLG-CTAB-DNA treated BMDCs, 14)pCMV-gag DNA PCR positive control. As illustrated in FIG. 1, the geneproduct was only detected by RT-PCR in PLG-CTAB-DNA preparations, andwas not the result of plasmid DNA contamination in the mRNA preparationas shown by the control PCR-only reactions. Hence, PLG-CTAB-DNAmicroparticles facilitate gene expression in BMDC. It is of interest tonote, however, that unsuccessful attempts were made to detect reportergene products, both luciferase and (3-galactosidase, in BMDC celllysates by luminometer and colorimetric substrate respectively.

Example 3

Stimulation of T cells. Bone marrow cells differentiated in the presenceof GM-CSF for 6 days were classified as immature as determined by FACSanalysis of cell surface phenotype CD11c⁺, CD11b⁺, H-dK^(d+),I-A^(d(low)), CD80^((low)), and CD86^((low)), and mature by day 9(CD11c⁺, CD11b⁺, H-2K^(d+), I-A^(d((bright)), CD80⁺, CD86⁺) (R. C.Fields, J. J. O., J. A. Fuller, E. K. Thomas, P. J. Geraghty, and J. J.Mule'. 1998. Comparative analysis of murine dendritic cells derived fromspleen and bone marrow. J. Immunother. 21:323). Both immature and matureBMDCs were stimulated for 24 h with PLG-CTAB-pCMVgag DNA or nakedpCMVgag DNA. Controls included untreated cells, microparticles alone orformulated with non-specific plasmid DNA (pCMV-luciferase) as well asnon-specific naked DNA. T cell hybridoma 12.2 (a d-restricted T cellhybridoma specific for the p7g epitope (AMQMLKETI) of HIV p55 gag) wasplated at 1×10⁵ cells per well of a 96 well, U-bottom microtiter plates.Varying numbers of BMDCs were plated with the hybridoma in a totalculture volume of 200 μl. Each individual experiment was performed induplicate. After a 24 h culture period, the plates were centrifuged andthe supernatants were removed and stored at

−80° C. until further assay for IL-2 production. To assay for levels ofIL-2 secreted into the medium, culture supernatants were thawed at roomtemperature and plated on pre-treated mouse IL-2 ELISA microtiter platesand analyzed per manufacturer's instructions (Endogen). Followingdevelopment of colorimetric substrate, microtiter plates were read by aMolecular Devices vmax kinetic plate reader and analyzed with SoftMaxsoftware.

As shown in FIG. 2, only PLG-CTAB-pCMV-gag treated BMDCs stimulatedlevels of IL-2 production above background. It is interesting to notethat immature cells thought to be efficient at antigen internalizationresulted in IL-2 levels that were 55% greater than background levelswhereas more mature BMDCs, which express higher levels of MHC moleculeson their cell surfaces, and are believed to be more efficient at antigenpresentation resulted in IL-2 levels 77% greater than background.Furthermore, PLG-CTAB-DNA-mediated stimulation of IL-2 production isdependent on the presence of antigen presenting cells, as the hybridomaalone treated with PLG-CTAB-DNA did not result in detectable levels ofIL-2 as determined by ELISA. Naked DNA in the presence of free CTAB alsodid not result in antigen presentation. Although PLG-CTAB-DNA treatmentresulted in transfection of dendritic cells in vitro, IL-2 productionwas two orders of magnitude less than that observed via a viraltechnique, i.e., with a recombinant vaccinia virus expressing the gaggene.

In addition to being antigen specific to a d-restricted epitope of theHIV p55gag antigen, the T cell hybridoma utilized in this study wasgenerated using the lacZ-inducible BWZ.36 fusion partner (provided by N.Shastri, U. of California Berkeley) which contains the Escherichia colilacZ reporter gene under the control of the nuclear factor of activatedT cells (NFAT) enhancer element of the IL-2 gene (Shastri, S. S. et al.1994. LacZ inducible, antigen/MHC-specific T cell hybrids. Intl.Immunol. 6:396). To confirm the results obtained by IL-2 ELISA, we alsoassayed the hybridoma cells by colorimetric assay for B-galactosidase(β-galactosidase staining kit, Invitrogen). Representative cell countsfrom microscope fields of view indicate a significant increase ofblue-stained cells over background in PLG-CTAB-pCMVgag-treated BMDCs(average counts 29 vs. 128 respectively).

The stimulation of IL-2 production by T cell recognition of antigenpresented in the context of MHC class I molecules was found to betime-dependent. Experiments (data not included) have shown that levelsof IL-2 production eventually decrease over time. However, significantlevels of IL-2 are produced after 7 days (about 15% of the 24-hour IL-2production level).

The stimulation of IL-2 production by T cell recognition of antigenpresented in the context of MHC class I molecules was found to bedose-dependent. In FIG. 3, levels of IL-2 production increase with thedose presented to BMDCs; however it is of interest to note thecorresponding increase in toxicity (lower % viability) that is alsoobserved. However, this deleterious effect may be abrogated by theapparent adjuvant activity of PLG-CTAB-DNA formulation.

As shown in FIG. 4, naïve BMDCs and BMDCs treated with PLG-CTAB-pCMVgagDNA were pulsed with an excess of synthetic p7g peptide epitope (1ng/ml) and serially diluted and plated with 1×10⁵ gag-specific MHC classI T hybridoma cells. Hence, various T cell to antigen presenting cellratios were provided, with the number of T cells being held constant.Stimulation was determined by IL-2 ELISA. As seen in FIG. 4, suchtreated cells become more efficient at T cell stimulation than untreatedcells. Stimulation of IL-2 production by the T cell hybridoma was dosedependent and detectable down to a T:APC ratio of 10000:1. Althoughpulsing surface MHC class I molecules with synthetic peptide epitope wasshown to be highly efficient at stimulating the T cell response, even inuntreated BMDCs, this is not expected to be a feasible approach togenetic immunotherapy due to the polymorphism of MHC class I epitopes inan outbred population. These data do however demonstrate upregulation ofMHC class I on the dendritic cell surface, a partial indication ofactivation by the PLG/CTAB formulation. This activation is expected tosignificantly increase the effectiveness of passively transferreddendritic cells transfected by the process of the invention.

As seen from the above, PLG-CTAB-DNA microparticles can be efficientlyinternalized by dendritic cells. Without wishing to be held to anyparticular theory, the presence of the cationic surfactants on thesurface may contribute to endosome disruption and cytoplasmic or nuclearlocalization. Gene expression was also observed by reverse-transcriptasePCR, indicating direct transfection of BMDCs in vitro. To exploit thepotent antigen uptake and presentation capabilities of dendritic cells,it was of interest to determine whether expressed antigen can beprocessed and presented on MHC molecules. It was seen that BMDCsincubated with PLG-CTAB microparticles formulated with pCMVgag plasmidencoding the HIV gag protein specifically stimulate antigen-specific Tcell hybridoma, resulting in the production of IL-2. Moreover, it hasbeen shown that such microparticles allow greater transfection thanunmodified plasmid DNA, using the T cell hybridoma-based readout. It hasalso been demonstrated that pulsing of dendritic cells with PLG-CTAB-DNAis an effective mechanism for rapid internalization, target geneexpression, and antigen presentation in vitro.

Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A method of transfecting dendritic cells comprising: incubatingdendritic cells and a transfection agent that comprises apolynucleotide, which encodes an antigen associated with a virus, abacterium, a parasite, a fungus or a tumor, adsorbed on surfaces ofmicroparticles, said incubating being performed ex vivo, for a timesufficient to transfect the dendritic cells with the polynucleotide,thereby leading to the expression of said antigen, wherein saidmicroparticles comprise a biodegradable polymer and a cationicdetergent, and wherein polynucleotide is not entrapped within saidmicroparticles.
 2. The method of claim 1, wherein the dendritic cellsoriginate from bone marrow.
 3. The method of claim 1, wherein thedendritic cells originate from blood.
 4. The method of claim 1, whereinthe dendritic cells originate from a vertebrate subject.
 5. The methodof claim 1, wherein the dendritic cells originate from a human subject.6. The method of claim 1, wherein the cationic detergent is cetyltrimethyl ammonium bromide.
 7. The method of claim 1, wherein thecationic detergent is cetrimide.
 8. The method of claim 1, wherein thepolymer is a poly(α-hydroxy acid).
 9. The method of claim 1, wherein thepolymer is a poly(lactide).
 10. The method of claim 1, wherein thepolymer is a poly(D,L-lactide-co-glycolide).
 11. The method of claim 1,wherein the polymer is a copolymer of D,L-lactide and caprolactone. 12.The method of claim 1, wherein the dendritic cells are cultured forabout 5 days prior to transfection.
 13. The method of claim 1, whereinthe dendritic cells are cultured for about 5 to about 10 days prior totransfection.
 14. The method of claim 1, wherein the dendritic cells andtransfecting agent are incubated for about 24 hours.
 15. The method ofclaim 1, wherein said polynucleotide is provided in the form of aplasmid.
 16. The method of claim 1, wherein the antigen is associatedwith human immunodeficiency virus, herpes simplex virus, hepatitis Bvirus, hepatitis C virus, human papillomavirus, influenza A virus,meningitis A, meningitis B, or meningitis C.
 17. A method for producingan immune response comprising administering, to a vertebrate subject inneed thereof, an effective amount of dendritic cells produced by themethod of claim
 1. 18. The method according to claim 17, in which thedendritic cells originate from the vertebrate subject.
 19. The methodaccording to claim 17, in which the dendritic cells originate from ahealthy vertebrate subject MHC-matched to the vertebrate subject. 20.The method according to claim 17, in which the dendritic cells areadministered parenterally.
 21. The method according to claim 17, inwhich the dendritic cells are administered by direct injection intoaffected tissue.
 22. The method according to claim 1, wherein saidmicroparticles have diameters ranging from about 500 nm to about 30 μm.23. The method according to claim 1, wherein said transfection agentcontains on the order of 1% w/w polynucleotide.
 24. The method of claim1, wherein said polynucleotide encodes a viral antigen.
 25. The methodof claim 1, wherein said polynucleotide encodes a tumor antigen.
 26. Themethod of claim 1, wherein said polynucleotide encodes a bacterialantigen.
 27. The method of claim 1, wherein said polynucleotide encodesa parasitic antigen.
 28. The method of claim 1, wherein saidpolynucleotide encodes a fungal antigen.
 29. The method of claim 17,wherein said polynucleotide encodes a viral antigen.
 30. The method ofclaim 17, wherein said polynucleotide encodes a tumor antigen.
 31. Themethod of claim 17, wherein said polynucleotide encodes a bacterialantigen.
 32. The method of claim 17, wherein said polynucleotide encodesa parasitic antigen.
 33. The method of claim 17, wherein saidpolynucleotide encodes a fungal antigen.
 34. The method of claim 17,wherein said polynucleotide encodes a human immunodeficiency virusantigen, a herpes simplex virus antigen, a hepatitis B virus antigen, ahepatitis C virus antigen, a human papillomavirus antigen, an influenzaA virus antigen, a meningitis A antigen, a meningitis B antigen, or ameningitis C antigen.
 35. The method of claim 17, wherein the detergentis cetyl trimethyl ammonium bromide.
 36. The method of any one of claims2-7, 12-15, 16-21, 22-23, 24-35, and 1, wherein the polymer is apoly(lactide-co-glycolide).
 37. The method of any one of claims 2-9,10-14, 17-21, 22-23, 35 and 1, wherein the polynucleotide is anexpression vector encoding an antigen associated with a virus, abacterium, a parasite, a fungus or a tumor.
 38. The method of claim 1,wherein said microparticles are manufactured in the presence of saidcationic detergent.
 39. The method of claim 1, wherein saidmicroparticles comprise a biodegradable polymer selected frompolylactide and poly(lactide-co-glycolide).
 40. A method of transfectingdendritic cells comprising: providing dendritic cells; providing atransfection agent comprising polynucleotide adsorbed on surfaces ofmicroparticles, said transfection agent being formed by a process thatcomprises: (a) providing microparticles comprising a biodegradablepolymer and a cationic detergent, and (b) exposing said microparticlesto said polynucleotide, said polynucleotide encoding an antigenassociated with a virus, a bacterium, a parasite, a fungus or a tumor;and incubating the dendritic cells and the transfection agent ex vivofor a time sufficient to transfect the dendritic cells with thepolynucleotide, thereby leading to the expression of said antigen,wherein polynucleotide is not entrapped within said microparticles. 41.The method of claim 40, wherein said microparticles comprise abiodegradable polymer selected from polylactide andpoly(lactide-co-glycolide).
 42. The method of claim 1, wherein saiddendritic cells are mature dendritic cells.